The phosphorylations of myosin heavy chains (MHC) by protein kinase C and myosin light chains by protein kinase C and Ca2+-dependent myosin light chain kinase are known to be temporally correlated with Ca2+- and protein kinase C-dependent secretion of granules when RBL-2H3 cells are stimulated with antigen and other secretagogues. Also, these and other studies in which these phosphorylations were selectively blocked or induced by chemical agents support the notion that release of secretory granules requires phosphorylation by both kinases. We now find that RBL- 2H3 cells express exclusively the A isoform of MHC by Northern blot, reverse-transcriptase polymerase chain reaction, and Western blot. Unlike rat PC12 cells, as well as a wide variety of other cultured cells and tissues, MHC-B mRNA and protein were not detectable in RBL-2H3 cells. The deduced amino acid sequence (1961 amino acids) of MHC-A from cDNA clones of RBL-2H3 cells indicated high homology with rat, human and chicken cellular MHC-A especially in the domains that allow binding of ATP and actin. The amino acid sequence of non-muscle MHC-A from rat was 96% identical to that in human and 92% identical to that in chicken. Northern blot analysis of mRNA indicated the presence of single message of 7.4 kilobase (kb). Because RBL-2H3 cells can be stimulated to release secretory granules as well as newly generated arachidonic acid and cytokines but lack MHC-B, this cell line may provide a unique model to study the role of MHC-A specifically in cellular responses to antigen and other stimulants.